Antibody Applications

Antibody Applications Overview

Rockland Immunochemicals produces antibody reagents that can be used in various immunoassays. Immunoassays are typically applications where antibodies are used to measure the presence or concentration of analytes. Analytes can be macromolecules, drugs, toxins or small molecules. Macromolecules are often proteins, sometimes antibodies themselves, but may also be lipids, carbohydrates, nucleic acids, whole cells or organisms.

Examples of Primary Antibodies Validated by Common Immunoassays

Antibody Protocols

Protocols are methods given for common immunoassays. Rockland makes available protocols for various immunoassays that can be used by research scientists in academic, private and government laboratories. It is recommended to follow these protocols without modification. In some instances, enzyme substrates, supporting reagents and buffers produced by Rockland are recommended for optimum results. Advanced users may modify procedures, however, it is recommended to change only one variable at a time in any given procedure. Primary antibodies and secondary antibodies should be validated for the intended immunoassay. The quality of water used in experiments is also of critical importance for best results. Always include positive and negative controls in immunoassays to interpret results correctly.

An analytical technique that combines the separation of proteins by gel electrophoresis followed by transfer to a membrane (typically nitrocellulose or PVDF) and immunodetection using specific antibodies. Often western blotting kits are used to facilitate research. Western blotting may be combined with immunoprecipitation using specialized detection antibodies such as TrueBlot® reagents.

A laser based technology that is used to count cells, cell sorting and to detect biomarker proteins that marks cells suspended in a stream of fluid using fluorescence followed by passing through a detector.

IHC is a process to detect antigens in cells or tissues using specific antibodies. Some antibody-antigen combinations require a process referred to as antigen retrieval to make the antigens more accessible for reaction with antibodies.

Gel Shift Assay

Also known as electrophorectic mobility shift assay (EMSA), or mobility shift electrophoresis, is an affinity electrophoresis technique used to study protein-DNA or protein –RNA interactions. When used in combination with a specific antibody for the detection of the protein component, an even greater shift in mobility is observed which is referred to as a gel supershift assay.

IP is the technique of precipitating an antigen from solution using a specific antibody that binds to the antigen of interest. Immunoprecipitation is often used to purify an antigen from a complex mixture of proteins. The precipitating antibody may be conjugated to a solid phase such as an agarose bead (i.e. antibody-agarose conjugates), or performed in solution where centrifugation is used to collect the antibody-antigen complex. Sepharose™ Protein A or Protein G conjugates are also used for immunoprecipitation. IP may be combined with western blotting using TrueBlot® reagents.

Neutralization Assay

A neutralization assay (NA) is an immunoassay whereby an antibody blocks a biological process. Neutralization assays include assays where antibodies block the ability of pathogens, including bacteria and viruses, from entering cells. For validated neutralizing primary antibodies click here. For a neutralization assay method click here.

Peptide Competition Assay

The peptide competition assay (PCA) is a recommended procedure for confirming the specific band reactivity of anti-peptide polyclonal antibodies, especially domain specific antibodies like phospho-specific antibodies. It is not uncommon to see more than one band on immunoblots of cell lysates when probing with a primary antibody. The PCA provides a means of determining which band or staining pattern is specific for the antibody.

Polyacrylamide gel electrophoresis in the presence of the detergent SDS is a widely used technique to separate complex mixtures of proteins according to their electrophoretic mobility. For SDS-PAGE, mobility is a function of length of the protein and is independent of charge or conformation. SDS-PAGE must be performed using molecular weight standards which are often prestained for visualization. SDS-PAGE buffers must be carefully prepared to obtain optimum results.

Chromatin Immunoprecipitation

ChIP is a specialized Immunoprecipitation technique used to investigate the binding of proteins to DNA within the cell. ChIP is used to study promoters, transcription factors and other DNA binding proteins including histones and modified histones. The field of epigenetics has benefited greatly from ChIP immunoassays. For a chromatin immunoprecipitation (ChIP) method click here.

The Rockland Advantage

Rockland validates primary antibodies by multiple immunoassays to ensure the highest quality performance of reagents. It is recommended whenever possible that the end user replicates experimental conditions used by Rockland during the validation process. Secondary antibodies, controls, standards, buffers and supporting reagents described in validation experiments should be used without deviation whenever possible to obtain superior results.